The detection of specific DNA target sequences present in air, food, water, environment or clinical samples is of great significance in the medical, veterinary, microbiology, food and water safety-testing, and environment monitoring fields. The detection for the presence of a specific genetic sequence in a sample can rapidly and correctly identify genetic defects, oncogenic events and bacterial, viral or parasitic agents of concern.
In the field of diagnostics of infectious diseases accurate identification of the causative organism is time-critical. Some rapid, sensitive and specific methods have been developed to replace the time-consuming and limited culture methods.
Diseases transmitted by blood-feeding vectors (parasites) are a growing threat to world health, particularly those affecting pets and transmitted by fleas, ticks, sandflies or mosquitoes (Vector Borne Diseases, VBD). VBD are known by worldwide veterinarians and usually have zoonotic consequences being important for human health.
Pets usually have a main role in the maintenance of zoonotic infections. Major VBD are provoked by Leishmania, Babesia, Anaplasma, Ehrlichia, Dirofilaria, Borrelia (Lyme disease), Rickettsia and Hepatozoon. Several of these are global infections and may cause severe diseases in humans. Pets may play an important role as parasite reservoirs and as sentinels of the infection due to their contact with humans.
One of the most important vector-borne diseases is visceral leishmaniasis which is endemic in 88 countries on 4 continents. The overall prevalence is 12 million people although the population at risk is 350 million. Leishmaniasis is regarded as the most important zoonotic disease within Europe. Zoonotic visceral leishmaniasis, caused by the protozoan Leishmania infantum and transmitted by sandfly vectors, is a fatal disease of domestic dogs, wild canids and humans which occurs mainly in the Mediterranean, Middle East and South America. The domestic dog is considered the major host for zoonotic leishmaniasis mainly in Europe. Moreover, leishmaniasis is emerging within non endemic areas and is thus increasingly becoming an important concern both from human public health and animal welfare perspectives due to movement of infected dogs, ongoing climatic change and the consequent extension of the range of the sandfly vector towards more northern latitudes.
Guidelines for canine Leishmaniasis diagnostic include clinical signs, clinicopathological abnormalities and serological status being molecular test of limited use for the need of skilled workers, the high cost and the fact that samples must be send to a reference lab. Moreover, the presence of lower antibody levels is not necessarily indicative of disease and further work-up is necessary to confirm it by other diagnostic methods, being PCR the gold standard.
Several molecular tests capable of direct parasite detection in an inexpensive, quantitative, reliable and friendly to use format to be performed at the clinician (point-of-care, POC) have been developed.
A dipstick format was developed for detection of PCR products by hybridization with a gold-conjugate probe, and a PCR oligochromatographic leishmania-specific test is being commercialized using this technology (Leishmania OligoC-test, Coris Bioconcept) (Deborggraeve S. et al. 2008. JID, 198:1565-1572). In 2011, the US Food and Drug Administration (FDA) approved the Smart Leish PCR assay (Cepheid USA), a qualitative real-time PCR test for diagnosing individuals with cutaneous leishmaniasis. Both systems did not overcome the limitations of PCR for which sophisticated and expensive equipment is needed to perform the precise and repeated heating cycles required.
Mohan (Mohan S. et al. 2011. Analyst, 136: 2845-2851) discloses an electrochemical detection assay for L. donovani wherein the genomic DNA of Leishmania is extracted and hybridized with a single strand oligonucleotide specific for L. donovani that is immobilized onto an ITO/NiO electrode. However, this method has low sensitivity and does not allow detecting double-stranded PCR amplicons.
Sabaté del Rio J. et al. (Sabaté del Rio J. et al. 2014. Biosensors and Bioelectronics, 54:674-678) disclose an electrochemical detection assay for Francisella tularensis. However, one of the limitations of this method is the lack of multiplex detection. Furthermore, it requires a DNA hybridization step which increases the analysis time and involves more irreproducibility, loss of sensitivity and false positives due to unspecific absorptions.
Therefore, there is still a need for further POC diagnostic methods for detecting infections by pathogens, particularly for diagnosing leishmaniasis.